Drug-Conjugates, Conjugation Methods, and Uses Thereof

ABSTRACT

In one aspect, an active agent-conjugate, methods of preparing the active agent-conjugate, and uses thereof is provided.

BACKGROUND

Recently, it has been found that an antibody (or antibody fragment suchas a single-chain variable fragment) can be linked to a payload drug toform an immunoconjugate that has been termed antibody-drug conjugate, orADC. The antibody causes the ADC to bind to the target cells. Often theADC is then internalized by the cell and the drug is released to treatthe cell. Because of the targeting, the side effects may be lower thanthe side effects of systemically administering the drug.

SUMMARY

Some embodiments provide active agent-conjugates, methods of preparingactive agent-conjugates, and uses thereof.

Some embodiments provide an active agent-conjugate having the structureof Formula I:

or a pharmaceutically acceptable salt thereof,wherein:A may be a targeting moiety;B may be an auxiliary moiety that optionally is a second targetingmoiety, or B may be null;L¹ includes a 2- to 5-carbon bridge and at least one sulfur atom;each D may be independently selected, where each D includes an activeagent;each L² may be independently a linker, wherein at least one L² links toL¹; andn may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

Some embodiments provide an active agent-conjugate having the structureof Formula Ia:

or a pharmaceutically acceptable salt thereof,wherein:the A-component may be a targeting moiety;the E-component may be an optionally substituted heteroaryl or anoptionally substituted heterocyclyl;B may be an auxiliary moiety that optionally is a second targetingmoiety, or B may be null;each D may be independently selected, where each D includes an activeagent;each L² may be independently a linker, wherein at least one L² links toL¹; andn may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;L³ may be an optionally substituted C₁-C₆ alkyl, or L³ may be null, whenL³ is null the sulfur is directly connected to the E-component; andL⁴ may be an optionally substituted C₁-C₆ alkyl, or L⁴ may be null, whenL⁴ is null the sulfur is directly connected to the E-component.

In some embodiments, the E-component includes a fragment selected fromthe group consisting of:

In some embodiments, L³ may be —(CH₂)—; and L⁴ may be —(CH₂)—. In someembodiments, L³ may be null; and L⁴ may be null.

In some embodiments,

may be:

In some embodiments, L² includes —(CH₂)_(n)— where n may be 1, 2, 3, 4,5, 6, 7, 8, 9, or 10. In some embodiments, L² includes —(CH₂CH₂O)_(n)—where n may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, L²includes Val-Cit-PAB, Val-Ala-PAB, Val-Lys(Ac)-PAB, Phe-Lys-PAB,Phe-Lys(Ac)-PAB, D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn-PAB, Ala-PAB,or PAB. In some embodiments, L² includes peptide, oligosaccharide,—(CH₂)_(n)—, —(CH₂CH₂O)_(n)—, Val-Cit-PAB, Val-Ala-PAB, Val-Lys(Ac)-PAB,Phe-Lys-PAB, Phe-Lys(Ac)-PAB, D-Val-Leu-Lys, Gly-Gly-Arg,Ala-Ala-Asn-PAB, Ala-PAB, PAB, or combinations thereof. In someembodiments, L² includes a noncleavable unit. In some embodiments, thenoncleavable unit includes —(CH₂)_(n)— where n may be 1, 2, 3, 4, 5, 6,7, 8, 9, or 10. In some embodiments, the noncleavable unit includes—(CH₂CH₂O)_(n)— where n may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In someembodiments, L² includes a cleavable unit. In some embodiments, thecleavable unit comprises a peptide.

In some embodiments, the A component comprises a monoclonal antibody(mAB). In some embodiments, the A component comprises an antibodyfragment, surrogate, or variant. In some embodiments, the A componentcomprises a protein ligand. In some embodiments, the A componentcomprises a protein scaffold. In some embodiments, the A componentcomprises a peptide. In some embodiments, the A component comprises asmall molecule ligand. In some embodiments, the A component comprises atleast one modified L-Alanine residue. In some embodiments, the Acomponent comprises at least two modified L-Alanine residues. In someembodiments, at least one L² includes —(CH₂)_(n)— where n may be 1, 2,3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, at least one L²includes —(CH₂CH₂O)_(n)— where n may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or10. In some embodiments, at least one L² includes Val-Cit-PAB,Val-Ala-PAB, Val-Lys(Ac)-PAB, Phe-Lys-PAB, Phe-Lys(Ac)-PAB,D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn-PAB, Ala-PAB, or PAB. In someembodiments, at least one L² includes a peptide, an oligosaccharide,—(CH₂)_(n)—, —(CH₂CH₂O)_(n)—, Val-Cit-PAB, Val-Ala-PAB, Val-Lys(Ac)-PAB,Phe-Lys-PAB, Phe-Lys(Ac)-PAB, D-Val-Leu-Lys, Gly-Gly-Arg,Ala-Ala-Asn-PAB, Ala-PAB, PAB or combinations thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the HIC-HPLC chromatogram product of reacting drug andantibody at a drug/antibody ratio of 5.5:1.

FIG. 2 shows the HIC-HPLC chromatogram product of reacting drug andantibody at a drug/antibody ratio of 6:1.

FIG. 3 shows the HIC-HPLC chromatogram product of reacting drug andantibody at a drug/antibody ratio of 1:6.5.

FIG. 4 shows the HIC-HPLC chromatogram product of reacting drug andantibody at a drug/antibody ratio of 1:4

FIG. 5 shows HIC-HPLC chromatograms for each FPLC fraction, and, inset,an overlay of the peaks to demonstrate relative purity of each peak.

FIG. 6 shows reduced SDS-PAGE analysis for the FPLC fractions depictedin FIGS. 4 and 5.

FIG. 7 shows viability of SK-BR-3 cells for seven antibody-drugconjugate samples.

FIG. 8 shows viability of HCC1954 cells for seven antibody-drugconjugate samples.

FIG. 9 shows reduced SDS-PAGE analysis for trastuzumab conjugatedcompound 40.

FIG. 10 shows viability of SK-BR-3 cells for four antibody-drugconjugate samples.

FIG. 11 shows viability of HCC1954 cells for four antibody-drugconjugate samples.

FIG. 12 shows viability of SK-BR-3 cells for four antibody-drugconjugate samples.

DETAILED DESCRIPTION

Some embodiments provide an active agent-conjugate. In some embodiments,the active agent-conjugate is a drug-conjugate. In some embodiments, thedrug-conjugate includes a targeting molecule. In some embodiments, thetargeting molecule includes a monoclonal antibody (mAB). In someembodiments, the drug-conjugate includes a spacer or a multifunctionallinker. In some embodiments, the spacer connects to the mAB by a sulfidebond. In some embodiments, the multifunctional linker connects to themAB by a sulfide bond. In some embodiments, the spacer ormultifunctional linker may be optionally connected to an auxiliarymoiety. In some embodiments, the auxiliary moiety may be a secondtargeting molecule such as mAB and peptide. In some embodiments, theauxiliary moiety may be a hydrophilic polymer such as polyethyleneglycol (PEG), and the like. In some embodiments, the spacer ormultifunctional linker may include a 2- to 5-atom bridge. In someembodiments, the spacer or multifunctional linker may include a 4C (fourcarbon) bridge.

Conjugation methods to derivatize a polypeptide with a payload can beaccomplished using a maleimido or vinyl moiety which can react withindividual sulfhydryl group on an antibody via Michael additionreaction. The free sulfhydryl group can be formed by reducing adisulfide bond in an antibody. However, the structural integrity of anantibody can be compromised after opening disulfide bonds and attachingpayloads to the exposed free thiols. The compositions and methodsprovided herein provide conjugation through cysteine without decreasedstructural stability.

DEFINITIONS

As used herein, common organic abbreviations are defined as follows:

Ac Acetyl

aq. AqueousBOC or Boc tert-ButoxycarbonylBrOP bromo tris(dimethylamino) phosphonium hexafluorophosphateBu n-Butyl° C. Temperature in degrees CentigradeDCM methylene chloride

DEPC Diethylcyanophosphonate

DIC diisopropylcarbodiimide

DIEA Diisopropylethylamine DMA N,N′-Dimethylacetamide DMFN,N′-Dimethylformamide DTT Dithiothreitol

EDC 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide

Et Ethyl

EtOAc Ethyl acetate

Eq Equivalents Fmoc 9-Fluorenylmethoxycarbonyl g Gram(s)

h Hour (hours)HATU 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphateHOAt 1-Hydroxy-7-azabenzotriazole

HOBT N-Hydroxybenzotriazole

HPLC High-performance liquid chromatographyLC/MS Liquid chromatography-mass spectrometry

Me Methyl MeOH Methanol MeCN Acetonitrile mL Milliliter(s)

MS mass spectrometryPAB p-aminobenzylRP-HPLC reverse phase HPLCt-Bu tert-ButylTCEP Tris(2-carboxyethyl)phosphine

TEA Triethylamine

Tert, t tertiaryTFA Trifluoracetic acid

THF Tetrahydrofuran THP Tetrahydropyranyl

TLC Thin-layer chromatography

μL Microliter(s)

The term “pharmaceutically acceptable salt” refers to salts that retainthe biological effectiveness and properties of a compound and, which arenot biologically or otherwise undesirable for use in a pharmaceutical.In many cases, the compounds disclosed herein are capable of formingacid and/or base salts by virtue of the presence of amino and/orcarboxyl groups or groups similar thereto. Pharmaceutically acceptableacid addition salts can be formed with inorganic acids and organicacids. Inorganic acids from which salts can be derived include, forexample, hydrochloric acid, hydrobromic acid, sulfuric acid, nitricacid, phosphoric acid, and the like. Organic acids from which salts canbe derived include, for example, acetic acid, propionic acid, glycolicacid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinicacid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamicacid, mandelic acid, methanesulfonic acid, ethanesulfonic acid,p-toluenesulfonic acid, salicylic acid, and the like. Pharmaceuticallyacceptable base addition salts can be formed with inorganic and organicbases. Inorganic bases from which salts can be derived include, forexample, sodium, potassium, lithium, ammonium, calcium, magnesium, iron,zinc, copper, manganese, aluminum, and the like; particularly preferredare the ammonium, potassium, sodium, calcium and magnesium salts.Organic bases from which salts can be derived include, for example,primary, secondary, and tertiary amines, substituted amines includingnaturally occurring substituted amines, cyclic amines, basic ionexchange resins, and the like, specifically such as isopropylamine,trimethylamine, diethylamine, triethylamine, tripropylamine, andethanolamine. Many such salts are known in the art, as described in WO87/05297, Johnston et al., published Sep. 11, 1987 (incorporated byreference herein in its entirety).

As used herein, “C_(a) to C_(b)” or “C_(a-b)” in which “a” and “b” areintegers refer to the number of carbon atoms in the specified group.That is, the group can contain from “a” to “b”, inclusive, carbon atoms.Thus, for example, a “C₁ to C₄ alkyl” or “C₁₋₄ alkyl” group refers toall alkyl groups having from 1 to 4 carbons, that is, CH₃—, CH₃CH₂—,CH₃CH₂CH₂—, (CH₃)₂CH—, CH₃CH₂CH₂CH₂—, CH₃CH₂CH(CH₃)— and (CH₃)₃C—.

The term “halogen” or “halo,” as used herein, means any one of theradio-stable atoms of column 7 of the Periodic Table of the Elements,e.g., fluorine, chlorine, bromine, or iodine, with fluorine and chlorinebeing preferred.

As used herein, “alkyl” refers to a straight or branched hydrocarbonchain that is fully saturated (i.e., contains no double or triplebonds). The alkyl group may have 1 to 20 carbon atoms (whenever itappears herein, a numerical range such as “1 to 20” refers to eachinteger in the given range; e.g., “1 to 20 carbon atoms” means that thealkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbonatoms, etc., up to and including 20 carbon atoms, although the presentdefinition also covers the occurrence of the term “alkyl” where nonumerical range is designated). The alkyl group may also be a mediumsize alkyl having 1 to 9 carbon atoms. The alkyl group could also be alower alkyl having 1 to 4 carbon atoms. The alkyl group may bedesignated as “C₁₋₄ alkyl” or similar designations. By way of exampleonly, “C₁₋₄ alkyl” indicates that there are one to four carbon atoms inthe alkyl chain, i.e., the alkyl chain is selected from the groupconsisting of methyl, ethyl, propyl, iso-propyl, n-butyl, iso-butyl,sec-butyl, and t-butyl. Typical alkyl groups include, but are in no waylimited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiarybutyl, pentyl, hexyl, and the like.

As used herein, “alkoxy” refers to the formula —OR wherein R is an alkylas is defined above, such as “C₁₋₉ alkoxy”, including but not limited tomethoxy, ethoxy, n-propoxy, 1-methylethoxy (isopropoxy), n-butoxy,iso-butoxy, sec-butoxy, and tert-butoxy, and the like.

As used herein, “alkylthio” refers to the formula —SR wherein R is analkyl as is defined above, such as “C₁₋₉ alkylthio” and the like,including but not limited to methylmercapto, ethylmercapto,n-propylmercapto, 1-methylethylmercapto (isopropylmercapto),n-butylmercapto, iso-butylmercapto, sec-butylmercapto,tert-butylmercapto, and the like.

As used herein, “alkenyl” refers to a straight or branched hydrocarbonchain containing one or more double bonds. The alkenyl group may have 2to 20 carbon atoms, although the present definition also covers theoccurrence of the term “alkenyl” where no numerical range is designated.The alkenyl group may also be a medium size alkenyl having 2 to 9 carbonatoms. The alkenyl group could also be a lower alkenyl having 2 to 4carbon atoms. The alkenyl group may be designated as “C₂₋₄ alkenyl” orsimilar designations. By way of example only, “C₂₋₄ alkenyl” indicatesthat there are two to four carbon atoms in the alkenyl chain, i.e., thealkenyl chain is selected from the group consisting of ethenyl,propen-1-yl, propen-2-yl, propen-3-yl, buten-1-yl, buten-2-yl,buten-3-yl, buten-4-yl, 1-methyl-propen-1-yl, 2-methyl-propen-1-yl,1-ethyl-ethen-1-yl, 2-methyl-propen-3-yl, buta-1,3-dienyl,buta-1,2,-dienyl, and buta-1,2-dien-4-yl. Typical alkenyl groupsinclude, but are in no way limited to, ethenyl, propenyl, butenyl,pentenyl, and hexenyl, and the like.

As used herein, “alkynyl” refers to a straight or branched hydrocarbonchain containing one or more triple bonds. The alkynyl group may have 2to 20 carbon atoms, although the present definition also covers theoccurrence of the term “alkynyl” where no numerical range is designated.The alkynyl group may also be a medium size alkynyl having 2 to 9 carbonatoms. The alkynyl group could also be a lower alkynyl having 2 to 4carbon atoms. The alkynyl group may be designated as “C₂₋₄ alkynyl” orsimilar designations. By way of example only, “C₂₋₄ alkynyl” indicatesthat there are two to four carbon atoms in the alkynyl chain, i.e., thealkynyl chain is selected from the group consisting of ethynyl,propyn-1-yl, propyn-2-yl, butyn-1-yl, butyn-3-yl, butyn-4-yl, and2-butynyl. Typical alkynyl groups include, but are in no way limited to,ethynyl, propynyl, butynyl, pentynyl, and hexynyl, and the like.

The term “aromatic” refers to a ring or ring system having a conjugatedpi electron system and includes both carbocyclic aromatic (e.g., phenyl)and heterocyclic aromatic groups (e.g., pyridine). The term includesmonocyclic or fused-ring polycyclic (i.e., rings which share adjacentpairs of atoms) groups provided that the entire ring system is aromatic.

As used herein, “aryloxy” and “arylthio” refers to RO— and RS—, in whichR is an aryl as is defined above, such as “C₆₋₁₀ aryloxy” or “C₆₋₁₀arylthio” and the like, including but not limited to phenyloxy.

An “aralkyl” or “arylalkyl” is an aryl group connected, as asubstituent, via an alkylene group, such as “C₇₋₁₄ aralkyl” and thelike, including but not limited to benzyl, 2-phenylethyl,3-phenylpropyl, and naphthylalkyl. In some cases, the alkylene group isa lower alkylene group (i.e., a C₁₋₄ alkylene group).

As used herein, “heteroaryl” refers to an aromatic ring or ring system(i.e., two or more fused rings that share two adjacent atoms) thatcontain(s) one or more heteroatoms, that is, an element other thancarbon, including but not limited to, nitrogen, oxygen and sulfur, inthe ring backbone. When the heteroaryl is a ring system, every ring inthe system is aromatic. The heteroaryl group may have 5-18 ring members(i.e., the number of atoms making up the ring backbone, including carbonatoms and heteroatoms), although the present definition also covers theoccurrence of the term “heteroaryl” where no numerical range isdesignated. In some embodiments, the heteroaryl group has 5 to 10 ringmembers or 5 to 7 ring members. The heteroaryl group may be designatedas “5-7 membered heteroaryl,” “5-10 membered heteroaryl,” or similardesignations. Examples of heteroaryl rings include, but are not limitedto, furyl, thienyl, phthalazinyl, pyrrolyl, oxazolyl, thiazolyl,imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, triazolyl,thiadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl,quinolinyl, isoquinlinyl, benzimidazolyl, benzoxazolyl, benzothiazolyl,indolyl, isoindolyl, and benzothienyl.

A “heteroaralkyl” or “heteroarylalkyl” is heteroaryl group connected, asa substituent, via an alkylene group. Examples include but are notlimited to 2-thienylmethyl, 3-thienylmethyl, furylmethyl, thienylethyl,pyrrolylalkyl, pyridylalkyl, isoxazollylalkyl, and imidazolylalkyl. Insome cases, the alkylene group is a lower alkylene group (i.e., a C₁₋₄alkylene group).

As used herein, “carbocyclyl” means a non-aromatic cyclic ring or ringsystem containing only carbon atoms in the ring system backbone. Whenthe carbocyclyl is a ring system, two or more rings may be joinedtogether in a fused, bridged or spiro-connected fashion. Carbocyclylsmay have any degree of saturation provided that at least one ring in aring system is not aromatic. Thus, carbocyclyls include cycloalkyls,cycloalkenyls, and cycloalkynyls. The carbocyclyl group may have 3 to 20carbon atoms, although the present definition also covers the occurrenceof the term “carbocyclyl” where no numerical range is designated. Thecarbocyclyl group may also be a medium size carbocyclyl having 3 to 10carbon atoms. The carbocyclyl group could also be a carbocyclyl having 3to 6 carbon atoms. The carbocyclyl group may be designated as “C₃₋₆carbocyclyl” or similar designations. Examples of carbocyclyl ringsinclude, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cyclohexenyl, 2,3-dihydro-indene, bicycle[2.2.2]octanyl,adamantyl, and spiro[4.4]nonanyl.

A “(carbocyclyl)alkyl” is a carbocyclyl group connected, as asubstituent, via an alkylene group, such as “C₄₋₁₀ (carbocyclyl)alkyl”and the like, including but not limited to, cyclopropylmethyl,cyclobutylmethyl, cyclopropylethyl, cyclopropylbutyl, cyclobutylethyl,cyclopropylisopropyl, cyclopentylmethyl, cyclopentylethyl,cyclohexylmethyl, cyclohexylethyl, cycloheptylmethyl, and the like. Insome cases, the alkylene group is a lower alkylene group.

As used herein, “cycloalkyl” means a fully saturated carbocyclyl ring orring system. Examples include cyclopropyl, cyclobutyl, cyclopentyl, andcyclohexyl.

As used herein, “cycloalkenyl” means a carbocyclyl ring or ring systemhaving at least one double bond, wherein no ring in the ring system isaromatic. An example is cyclohexenyl.

As used herein, “heterocyclyl” means a non-aromatic cyclic ring or ringsystem containing at least one heteroatom in the ring backbone.Heterocyclyls may be joined together in a fused, bridged orspiro-connected fashion. Heterocyclyls may have any degree of saturationprovided that at least one ring in the ring system is not aromatic. Theheteroatom(s) may be present in either a non-aromatic or aromatic ringin the ring system. The heterocyclyl group may have 3 to 20 ring members(i.e., the number of atoms making up the ring backbone, including carbonatoms and heteroatoms), although the present definition also covers theoccurrence of the term “heterocyclyl” where no numerical range isdesignated. The heterocyclyl group may also be a medium sizeheterocyclyl having 3 to 10 ring members. The heterocyclyl group couldalso be a heterocyclyl having 3 to 6 ring members. The heterocyclylgroup may be designated as “3-6 membered heterocyclyl” or similardesignations. In preferred six membered monocyclic heterocyclyls, theheteroatom(s) are selected from one up to three of O (oxygen), N(nitrogen) or S (sulfur), and in preferred five membered monocyclicheterocyclyls, the heteroatom(s) are selected from one or twoheteroatoms selected from O (oxygen), N (nitrogen), or S (sulfur).Examples of heterocyclyl rings include, but are not limited to,azepinyl, acridinyl, carbazolyl, cinnolinyl, dioxolanyl, imidazolinyl,imidazolidinyl, morpholinyl, oxiranyl, oxepanyl, thiepanyl, piperidinyl,piperazinyl, dioxopiperazinyl, pyrrolidinyl, pyrrolidonyl,pyrrolidionyl, 4-piperidonyl, pyrazolinyl, pyrazolidinyl, 1,3-dioxinyl,1,3-dioxanyl, 1,4-dioxinyl, 1,4-dioxanyl, 1,3-oxathianyl,1,4-oxathiinyl, 1,4-oxathianyl, 2H-1,2-oxazinyl, trioxanyl,hexahydro-1,3,5-triazinyl, 1,3-dioxolyl, 1,3-dioxolanyl, 1,3-dithiolyl,1,3-dithiolanyl, isoxazolinyl, isoxazolidinyl, oxazolinyl, oxazolidinyl,oxazolidinonyl, thiazolinyl, thiazolidinyl, 1,3-oxathiolanyl, indolinyl,isoindolinyl, tetrahydrofuranyl, tetrahydropyranyl,tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydro-1,4-thiazinyl,thiamorpholinyl, dihydrobenzofuranyl, benzimidazolidinyl, andtetrahydroquinoline.

A “(heterocyclyl)alkyl” is a heterocyclyl group connected, as asubstituent, via an alkylene group. Examples include, but are notlimited to, imidazolinylmethyl and indolinylethyl.

As used herein, “acyl” refers to —C(═O)R, wherein R is hydrogen, C₁₋₆alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl, C₆₋₁₀ aryl, 5-10membered heteroaryl, and 5-10 membered heterocyclyl, as defined herein.Non-limiting examples include formyl, acetyl, propanoyl, benzoyl, andacryl.

An “O-carboxy” group refers to a “—OC(═O)R” group in which R is selectedfrom hydrogen, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl,C₆₋₁₀ aryl, 5-10 membered heteroaryl, and 5-10 membered heterocyclyl, asdefined herein.

A “C-carboxy” group refers to a “—C(═O)OR” group in which R is selectedfrom hydrogen, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl,C₆₋₁₀ aryl, 5-10 membered heteroaryl, and 5-10 membered heterocyclyl, asdefined herein. A non-limiting example includes carboxyl (i.e.,—C(═O)OH).

A “cyano” group refers to a “—CN” group.

A “cyanato” group refers to an “—OCN” group.

An “isocyanato” group refers to a “—NCO” group.

A “thiocyanato” group refers to a “—SCN” group.

An “isothiocyanato” group refers to an “—NCS” group.

A “sulfinyl” group refers to an “—S(═O)R” group in which R is selectedfrom hydrogen, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl,C₆₋₁₀ aryl, 5-10 membered heteroaryl, and 5-10 membered heterocyclyl, asdefined herein.

A “sulfonyl” group refers to an “—SO₂R” group in which R is selectedfrom hydrogen, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl,C₆₋₁₀ aryl, 5-10 membered heteroaryl, and 5-10 membered heterocyclyl, asdefined herein.

An “S-sulfonamido” group refers to a “—SO₂NR_(A)R_(B)” group in whichR_(A) and R_(B) are each independently selected from hydrogen, C₁₋₆alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl, C₆₋₁₀ aryl, 5-10membered heteroaryl, and 5-10 membered heterocyclyl, as defined herein.

An “N-sulfonamido” group refers to a “—N(R_(A))SO₂R_(B)” group in whichR_(A) and R_(b) are each independently selected from hydrogen, C₁₋₆alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl, C₆₋₁₀ aryl, 5-10membered heteroaryl, and 5-10 membered heterocyclyl, as defined herein.

An “O-carbamyl” group refers to a “—OC(═O)NR_(A)R_(B)” group in whichR_(A) and R_(B) are each independently selected from hydrogen, C₁₋₆alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl, C₆₋₁₀ aryl, 5-10membered heteroaryl, and 5-10 membered heterocyclyl, as defined herein.

An “N-carbamyl” group refers to an “—N(R_(A))C(═O)OR_(B)” group in whichR_(A) and R_(B) are each independently selected from hydrogen, C₁₋₆alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl, C₆₋₁₀ aryl, 5-10membered heteroaryl, and 5-10 membered heterocyclyl, as defined herein.

An “O-thiocarbamyl” group refers to a “—OC(═S)NR_(A)R_(B)” group inwhich R_(A) and R_(B) are each independently selected from hydrogen,C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl, C₆₋₁₀ aryl,5-10 membered heteroaryl, and 5-10 membered heterocyclyl, as definedherein.

A “urea” group refers to a “—N(R_(A))C(═O)NR_(A)R_(B)” group in whichR_(A) and R_(B) are each independently selected from hydrogen, C₁₋₆alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl, C₆₋₁₀ aryl, 5-10membered heteroaryl, and 5-10 membered heterocyclyl, as defined herein.

An “N-thiocarbamyl” group refers to an “—N(R_(A))C(═S)OR_(B)” group inwhich R_(A) and R_(B) are each independently selected from hydrogen,C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl, C₆₋₁₀ aryl,5-10 membered heteroaryl, and 5-10 membered heterocyclyl, as definedherein.

A “C-amido” group refers to a “—C(═O)NR_(A)R_(B)” group in which R_(A)and R_(B) are each independently selected from hydrogen, C₁₋₆ alkyl,C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl, C₆₋₁₀ aryl, 5-10 memberedheteroaryl, and 5-10 membered heterocyclyl, as defined herein.

An “N-amido” group refers to a “—N(R_(A))C(═O)R_(B)” group in whichR_(A) and R_(B) are each independently selected from hydrogen, C₁₋₆alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl, C₆₋₁₀ aryl, 5-10membered heteroaryl, and 5-10 membered heterocyclyl, as defined herein.

An “amino” group refers to a “—NR_(A)R_(B)” group in which R_(A) andR_(B) are each independently selected from hydrogen, C₁₋₆ alkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl, C₆₋₁₀ aryl, 5-10 memberedheteroaryl, and 5-10 membered heterocyclyl, as defined herein. Anon-limiting example includes free amino (i.e., —NH₂).

An “aminoalkyl” group refers to an amino group connected via an alkylenegroup.

An “alkoxyalkyl” group refers to an alkoxy group connected via analkylene group, such as a “C₂₋₈ alkoxyalkyl” and the like.

As used herein, a substituted group is derived from the unsubstitutedparent group in which there has been an exchange of one or more hydrogenatoms for another atom or group. Unless otherwise indicated, when agroup is deemed to be “substituted,” it is meant that the group issubstituted with one or more substitutents independently selected fromC₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₇ carbocyclyl (optionallysubstituted with halo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, andC₁-C₆ haloalkoxy), C₃-C₇-carbocyclyl-C₁-C₆-alkyl (optionally substitutedwith halo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, and C₁-C₆haloalkoxy), 5-10 membered heterocyclyl (optionally substituted withhalo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, and C₁-C₆ haloalkoxy),5-10 membered heterocyclyl-C₁-C₆-alkyl (optionally substituted withhalo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, and C₁-C₆ haloalkoxy),aryl (optionally substituted with halo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆haloalkyl, and C₁-C₆ haloalkoxy), aryl(C₁-C₆)alkyl (optionallysubstituted with halo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, andC₁-C₆ haloalkoxy), 5-10 membered heteroaryl (optionally substituted withhalo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, and C₁-C₆ haloalkoxy),5-10 membered heteroaryl(C₁-C₆)alkyl (optionally substituted with halo,C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, and C₁-C₆ haloalkoxy), halo,cyano, hydroxy, C₁-C₆ alkoxy, C₁-C₆ alkoxy(C₁-C₆)alkyl (i.e., ether),aryloxy, sulfhydryl (mercapto), halo(C₁-C₆)alkyl (e.g., —CF₃),halo(C₁-C₆)alkoxy (e.g., —OCF₃), C₁-C₆ alkylthio, arylthio, amino,amino(C₁-C₆)alkyl, nitro, O-carbamyl, N-carbamyl, O-thiocarbamyl,N-thiocarbamyl, C-amido, N-amido, S-sulfonamido, N-sulfonamido,C-carboxy, O-carboxy, acyl, cyanato, isocyanato, thiocyanato,isothiocyanato, sulfinyl, sulfonyl, and oxo (═O). Wherever a group isdescribed as “optionally substituted” that group can be substituted withthe above substituents.

It is to be understood that certain radical naming conventions caninclude either a mono-radical or a di-radical, depending on the context.For example, where a substituent requires two points of attachment tothe rest of the molecule, it is understood that the substituent is adi-radical. For example, a substituent identified as alkyl that requirestwo points of attachment includes di-radicals such as —CH₂—, —CH₂CH₂—,—CH₂CH(CH₃)CH₂—, and the like. Other radical naming conventions clearlyindicate that the radical is a di-radical such as “alkylene” or“alkenylene.”

Wherever a substituent is depicted as a di-radical (i.e., has two pointsof attachment to the rest of the molecule), it is to be understood thatthe substituent can be attached in any directional configuration unlessotherwise indicated. Thus, for example, a substituent depicted as -AE-or

includes the substituent being oriented such that the A is attached atthe leftmost attachment point of the molecule as well as the case inwhich A is attached at the rightmost attachment point of the molecule.

“Subject” as used herein, means a human or a non-human mammal, e.g., adog, a cat, a mouse, a rat, a cow, a sheep, a pig, a goat, a non-humanprimate or a bird, e.g., a chicken, as well as any other vertebrate orinvertebrate.

Conjugation Methods, Spacers and Linkers Involved

Some embodiments provide a method of conjugating of a targeting moleculethrough a spacer or a multifunctional linker. In some embodiments, thespacer or multifunctional linker may include a 2- to 5-atom bridge. Insome embodiments, the method includes a single-step or sequentialconjugation approach. In some embodiments, the drug-conjugates include aspacer or a multifunctional linker. In some embodiments, the spacer ormultifunctional linker may include a noncleavable or cleavable unit suchas peptides.

Utilities and Applications

Some embodiments provide a method of treating a patient in need thereofcomprising administering an active agent-conjugate as disclosed anddescribed herein to said patient. In some embodiments, the patient mayhave cancer, immune diseases or diabetes.

Some embodiments provide a method of diagnosis or imaging comprisingadministering an active agent-conjugate as disclosed and describedherein to an individual.

Certain Structures

Some embodiments provide an active agent-conjugate having the structureof Formula I

or a pharmaceutically acceptable salt thereof,

wherein:

A may be a targeting molecule;

B is an auxiliary moiety that optionally is a second targeting molecule,or B is null;

L¹ includes a 2- to 5-carbon bridge and at least one sulfur atom;

each D is independently selected, where each D includes an active agent;

each L² is independently a linker, wherein at least one L² links to L¹;

and

n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In some embodiments, A may be a monoclonal antibody (mAB).

In some embodiments, A may be an antibody fragment, surrogate, orvariant.

In some embodiments, A may be a protein ligand.

In some embodiments, A may be a protein scaffold.

In some embodiments, A may be a peptide.

In some embodiments, A may be a small molecule ligand.

In some embodiments, B may be a hydrophilic polymer. In someembodiments, the hydrophilic polymer may polyethylene glycol (PEG), andthe like. In some embodiments, B may be a biodegradable polymer. In someembodiments, the biodegradable polymer may be unstructured proteinspolyamino acids, polypeptides polysaccharides and combinations thereof

In some embodiments, B may be a monoclonal antibody (mAB).

In some embodiments, B may be an antibody fragment, surrogate, orvariant.

In some embodiments, B may be a protein ligand.

In some embodiments, B may be a protein scaffold.

In some embodiments, B may be a peptide.

In some embodiments, B may be RNA or DNA.

In some embodiments, B may be a RNA or DNA fragment.

In some embodiments, B may be a small molecule ligand.

In some embodiments, D may be a biologically active compound.

In some embodiments, D may be a drug.

In some embodiments, D may be a chemotherapy drug.

In some embodiments, D may be a natural product.

In some embodiments, D may be an immune modulator.

In some embodiments, D may be a tubulin-binder.

In some embodiments, D may be a DNA-alkylating agent.

In some embodiments, D may be an HSP90 inhibitor.

In some embodiments, D may be a DNA topoisomerase inhibitor.

In some embodiments, D may be an anti-epigenetic agent.

In some embodiments, D may be an HDAC inhibitor.

In some embodiments, D may be an anti-metabolism agent.

In some embodiments, D may be a proteasome inhibitor.

In some embodiments, D may be a peptide.

In some embodiments, D may be a peptidomimetic.

In some embodiments, D may be an siRNA.

In some embodiments, D may be an antisense DNA.

In some embodiments, D may be epothilone A, epothilone B, or paclitaxel.

In some embodiments, L² may include a spacer or a multifunctionallinker. In some embodiments, L² may include a spacer and amultifunctional linker. In some embodiments, L² may include amultifunctional linker. In some embodiments, each L² may be a linker,wherein the linker may be cleavable or non-cleavable under biologicalconditions. In some embodiments, the linker may be cleavable by anenzyme. In some embodiments, L² may include Linker.

In some embodiments, L¹ includes a 2- to 5-carbon bridge and at leastone sulfur atom. In some embodiments, L¹ includes a 2- to 5-carbonbridge and at least two sulfur atoms. In some embodiments, L¹ includes a2- to 5-carbon bridge and a spacer. In some embodiments, L¹ includes a2- to 5-carbon bridge, at least two sulfur atoms and a spacer. In someembodiments, L¹ may include one or more sulfurs. In some embodiments,the L¹ may include two or more sulfurs. In some embodiments, the L¹ mayinclude exactly two sulfurs. In some embodiments, may include a 4-carbonbridge and/or a spacer. In some embodiments, L¹ include a 4-carbonbridge or a spacer. In some embodiments, L¹ may include a 4-carbonbridge and a spacer. In some embodiments, L¹ includes a 4-carbon bridgeand at least two sulfur atoms. In some embodiments, the spacer connectsto the mAB by a sulfide bond. In some embodiments, the spacer connectsto the mAB through a thioether.

In some embodiments, A comprises at least one modified L-Alanineresidue. In some embodiments, A comprises at least two modifiedL-Alanine residues. In some embodiments, L¹ comprises at least onesulfur. In some embodiments, L¹ comprises at least two sulfurs. In someembodiments, A comprises at least one modified L-Alanine residue that isconnected to at least one sulfur of L¹. In some embodiments, at leastone modified L-Alanine residue is from an L-Cysteine residue of apeptide before conjugation. In some embodiments, at least one sulfur ofL¹ is from an L-Cysteine of a peptide before conjugation. In someembodiments, A and L¹ together comprise at least one thioether.

In some embodiments, Linker may be a peptide.

In some embodiments, Linker may include an oligosaccharide. For example,Linker may include chitosan. In some embodiments, L² may include Linkerand —(CH₂)_(n)— where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In someembodiments, L² may include Linker and —(CH₂CH₂O)_(n)— where n is 1, 2,3, 4, 5, 6, 7, 8, 9, or 10.

In some embodiments, Linker may include —(CH₂)_(n)— where n is 1, 2, 3,4, 5, 6, 7, 8, 9, or 10.

In some embodiments, Linker may include —(CH₂CH₂O)_(n)— where n is 1, 2,3, 4, 5, 6, 7, 8, 9, or 10.

In some embodiments, Linker may include Val-Cit-PAB, Val-Ala-PAB,Val-Lys(Ac)-PAB, Phe-Lys-PAB, Phe-Lys(Ac)-PAB, D-Val-Leu-Lys,Gly-Gly-Arg, Ala-Ala-Asn-PAB, Ala-PAB, PAB, or the like.

In some embodiments, Linker may include any combination of peptide,oligosaccharide, —(CH₂)_(n)—, —(CH₂CH₂O)_(n)—, Val-Cit-PAB, Val-Ala-PAB,Val-Lys(Ac)-PAB, Phe-Lys-PAB, Phe-Lys(Ac)-PAB, D-Val-Leu-Lys,Gly-Gly-Arg, Ala-Ala-Asn-PAB, Ala-PAB, PAB, and the like.

In some embodiments, the spacer may include a peptide.

In some embodiments, the spacer may include an oligosaccharide. Forexample, the spacer may include chitosan.

In some embodiments, the spacer may include —(CH₂)_(n)— where n is 1, 2,3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, L¹ may include acomponent including a 4-carbon bridge and —(CH₂)_(n)— where n is 1, 2,3, 4, 5, 6, 7, 8, 9, or 10.

In some embodiments, the spacer may include —(CH₂CH₂O)_(n)— where n is1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, L¹ may include acomponent including a 4-carbon bridge and —(CH₂CH₂O)_(n)— where n is 1,2, 3, 4, 5, 6, 7, 8, 9, or 10.

In some embodiments, the spacer may include Val-Cit-PAB, Val-Ala-PAB,Val-Lys(Ac)-PAB, Phe-Lys-PAB, Phe-Lys(Ac)-PAB, D-Val-Leu-Lys,Gly-Gly-Arg, Ala-Ala-Asn-PAB, Ala-PAB, PAB, or the like.

In some embodiments, the spacer may be any combination of peptide,oligosaccharide, —(CH₂)_(n)—, —(CH₂CH₂O)_(n)—, Val-Cit-PAB, Val-Ala-PAB,Val-Lys(Ac)-PAB, Phe-Lys-PAB, Phe-Lys(Ac)-PAB, D-Val-Leu-Lys,Gly-Gly-Arg, Ala-Ala-Asn-PAB, Ala-PAB, PAB, and the like.

In some embodiments, L¹ may include a 4-carbon bridge. In someembodiments, L¹ may include a 4-carbon bridge through a fused aromaticring. In some embodiments, L¹ may include a spacer and componentincluding a 4-carbon bridge through a fused aromatic ring. In someembodiments, L¹ may include, but is not limited

and the like.

In some embodiments, the agent-conjugate having the structure of FormulaI has the structure of Formula Ia:

or a pharmaceutically acceptable salt thereof,wherein the S-linked portion of A comprises a modified L-Alanineresidue, wherein

may connect to a sulfur of a reduced disulfide bond through a bridgecontaining 2 to 5 atoms. For example, the structure indicated by

may include a fragment selected from the group consisting of:

In some embodiments, the S-linked (sulfur-linked) portion of A comprisesa modified L-Alanine residue. In some embodiments, the S-linked(sulfur-linked) portion of A comprises a modified L-Alanine residue thatis connected to the sulfur of L¹. In some embodiments, the S-linked(sulfur-linked) portion of A comprises a modified L-Alanine residuewherein the modified L-Alanine component of A is from an L-Cysteineresidue of a peptide before conjugation. In some embodiments, the sulfurof L¹ is from an L-Cysteine of a peptide before conjugation. In someembodiments, A and L¹ comprise a thioether. In some embodiments, thecompound having the structure of Formula Ia may be formed from a peptidethat includes at least one L-Cysteine where the L-Cysteine provides amodified L-Alanine of A and a sulfur of

In some embodiments, the compound having the structure of Formula Ia maybe formed from a peptide that includes at least two L-Cysteine where thetwo L-Cysteine provide two modified L-Alanines of A and two sulfur of

In some embodiments, the agent-conjugate having the structure of FormulaI has the structure of Formula Ib:

or a pharmaceutically acceptable salt thereof, wherein the A-componentmay be a targeting moiety, the E-component may be a heteroaryl orheterocyclyl, L³ may be an optionally substituted C₁-C₆ alkyl, or L³ maybe null, when L³ is null the sulfur is directly connected to theE-component; and L⁴ may be an optionally substituted C₁-C₆ alkyl, or L⁴may be null, when L⁴ is null the sulfur is directly connected to theE-component. In some embodiments, L³ may be —(CH₂)—; and L⁴ may be—(CH₂)—. In some embodiments, L³ may be null; and L⁴ may be null.

In some embodiments of the structure of Formula Ib, the structuralcomponent

may be:

In some embodiments, the E-component includes a fragment selected fromthe group consisting of:

In some embodiments, the active agent may be selected from the groupconsisting of tubulin binders, DNA alkylators, DNA intercalator, enzymeinhibitors, immune modulators, peptides, and nucleotides.

In some embodiments, at least one L² includes —(CH₂)_(n)— where n is 1,2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, at least one L²includes —(CH₂CH₂O)_(n)— where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Insome embodiments, at least one L² includes Val-Cit-PAB, Val-Ala-PAB,Val-Lys(Ac)-PAB, Phe-Lys-PAB, Phe-Lys(Ac)-PAB, D-Val-Leu-Lys,Gly-Gly-Arg, Ala-Ala-Asn-PAB, Ala-PAB, or PAB. In some embodiments, atleast one L² includes a peptide, an oligosaccharide, —(CH₂)_(n)—,—(CH₂CH₂O)_(n)—, Val-Cit-PAB, Val-Ala-PAB, Val-Lys(Ac)-PAB, Phe-Lys-PAB,Phe-Lys(Ac)-PAB, D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn-PAB, Ala-PAB,or PAB. In some embodiments, at least one L² may comprise, consist of,or consist essentially of:

In some embodiments, the agent-conjugate having the structure of FormulaI has the structure of

In some embodiments, the agent-conjugate having the structure of FormulaI has the structure of

In some embodiments, the agent-conjugate having the structure of FormulaI has the structure of

In some embodiments, the agent-conjugate having the structure of FormulaI has the structure of

In some embodiments, the agent-conjugate having the structure of FormulaI has the structure of

In some embodiments, the agent-conjugate having the structure of FormulaI has the structure of

In some embodiments, the agent-conjugate having the structure of FormulaI has the structure of

In some embodiments, the agent-conjugate having the structure of FormulaI has the structure of

In some embodiments, the agent-conjugate having the structure of FormulaI has the structure of

In some embodiments, the agent-conjugates may include one or morecomponents selected from the group consisting of an amino acid, an aminoacid residue, an amino acid analog, and a modified amino acid.

As used herein, the term “targeting moiety” refers to a structure thatbinds or associates with a biological moiety or fragment thereof

In some embodiments, the targeting moiety may be an antibody. In someembodiments, the targeting moiety may be a monoclonal antibody (mAB). Insome embodiments, the targeting moiety may be an antibody fragment,surrogate, or variant. In some embodiments, the targeting moiety may bea protein ligand. In some embodiments, the targeting moiety may be aprotein scaffold. In some embodiments, the targeting moiety may be apeptide. In some embodiments, the targeting moiety may be RNA or DNA. Insome embodiments, the targeting moiety may be a RNA or DNA fragment. Insome embodiments, the targeting moiety may be a small molecule ligand.

In some embodiments, the targeting moiety may be an antibody fragmentdescribed in Janthur et al., “Drug Conjugates Such as Antibody DrugConjugates (ADCs), Immunotoxins and Immunoliposomes Challenge DailyClinical Practice,” Int. J. Mol. Sci. 2012, 13, 16020-16045, thedisclosure of which is incorporated herein by reference in its entirety.In some embodiments, the targeting moiety may be an antibody fragmentdescribed in Trail, P A, “Antibody Drug Conjugates as CancerTherapeutics,” Antibodies 2013, 2, 113-129, the disclosure of which isincorporated herein by reference in its entirety.

In some embodiments, the targeting moiety may be HuM195-Ac-225,HuM195-Bi-213, Anyara (naptumomab estafenatox; ABR-217620), AS1409,Zevalin (ibritumomab tiuxetan), BIIB015, BT-062, Neuradiab, CDX-1307,CR011-vcMMAE, Trastuzumab-DM1 (R3502), Bexxar (tositumomab), IMGN242,IMGN388, IMGN901, ¹³¹I-labetuzumab, IMMU-102 (⁹⁰Y-epratuzumab), IMMU-107(⁹⁰Y-clivatuzumab tetraxetan), MDX-1203, CAT-8015, EMD 273063(hu14.18-IL2), Tucotuzumab celmoleukin (EMD 273066; huKS-IL2),¹⁸⁸Re-PTI-6D2, Cotara, L19-IL2, Teleukin (F16-IL2), Tenarad (F16-¹³¹I),L19-¹³¹I, L19-TNF, PSMA-ADC, DI-Leu16-IL2, SAR3419, SGN-35, or CMC544.In some embodiments, the targeting moiety may comprise, consist of, orconsist essentially of the antibody portion of HuM195-Ac-225,HuM195-Bi-213, Anyara (naptumomab estafenatox; ABR-217620), AS 1409,Zevalin (ibritumomab tiuxetan), BIIB015, BT-062, Neuradiab, CDX-1307,CR011-vcMMAE, Trastuzumab-DM1 (R3502), Bexxar (tositumomab), IMGN242,IMGN388, IMGN901, ¹³¹I-labetuzumab, IMMU-102 (⁹⁰Y-epratuzumab), IMMU-107(⁹⁰Y-clivatuzumab tetraxetan), MDX-1203, CAT-8015, EMD 273063(hu14.18-IL2), Tucotuzumab celmoleukin (EMD 273066; huKS-IL2),¹⁸⁸Re-PTI-6D2, Cotara, L19-IL2, Teleukin (F16-IL2), Tenarad (F16-¹³¹I),L19-¹³¹I, L19-TNF, PSMA-ADC, DI-Leu16-IL2, SAR3419, SGN-35, or CMC544.

In some embodiments, the targeting moiety may be Brentuximab vedotin,Trastuzumab emtansine, Inotuzumab ozogamicin, Lorvotuzumab mertansine,Glembatumumab vedotin, SAR3419, Moxetumomab pasudotox, Moxetumomabpasudotox, AGS-16M8F, AGS-16M8F, BIIB-015, BT-062, IMGN-388, orIMGN-388.

In some embodiments, the targeting moiety may comprise, consist of, orconsist essentially of the antibody portion of Brentuximab vedotin,Trastuzumab emtansine, Inotuzumab ozogamicin, Lorvotuzumab mertansine,Glembatumumab vedotin, SAR3419, Moxetumomab pasudotox, Moxetumomabpasudotox, AGS-16M8F, AGS-16M8F, BIIB-015, BT-062, IMGN-388, orIMGN-388.

In some embodiments, the targeting moiety may comprise, consist of, orconsist essentially of Brentuximab, Inotuzumab, Gemtuzumab, Milatuzumab,Trastuzumab, Glembatumomab, Lorvotuzumab, or Labestuzumab.

As used herein, the term “peptide” refers to a structure including oneor more components each individually selected from the group consistingof an amino acid, an amino acid residue, an amino acid analog, and amodified amino acid. The components are typically joined to each otherthrough an amide bond.

In some embodiments, the peptide, such as the antibody, is PEGylated.PEGylation can provide, for example, increased stability and/or efficacyof the polypeptide. Methods for PEGylation known in the art can be usedin the methods and compositions provided herein. Such methods include,but are not limited to, those provided in “Comparative Binding ofDisulfide-Bridged PEG-Fabs” Khalili et al., Bioconjugate Chemistry(2012), 23(11), 2262-2277; “Site-Specific PEGylation at Histidine Tags”Cong et al., Bioconjugate Chemistry (2012), 23(2), 248-263; “Disulfidebridge based PEGylation of proteins” Brocchini et al., Advanced DrugDelivery Reviews (2008), 60(1), 3-12; “Site-Specific PEGylation ofProtein Disulfide Bonds Using a Three-Carbon Bridge” Balan et al.,Bioconjugate Chemistry (2007), 18(1), 61-76; “Site-specific PEGylationof native disulfide bonds in therapeutic proteins” Shaunak et al.,Nature Chemical Biology (2006), 2(6), 312-313; “PEG derivativeconjugated proteins and peptides for use in pharmaceuticals” Godwin etal., WO 2010/100430. All of the aforementioned PEGylation references areincorporated by references in their entireties.

As used herein, the term “amino acid” includes naturally occurring aminoacids, a molecule having a nitrogen available for forming an amide bondand a carboxylic acid, a molecule of the general formula NH₂—CHR—COOH orthe residue within a peptide bearing the parent amino acid, where “R” isone of a number of different side chains. “R” can be a substituent foundin naturally occurring amino acids. “R” can also be a substituentreferring to one that is not of the naturally occurring amino acids.

As used herein, the term “amino acid residue” refers to the portion ofthe amino acid which remains after losing a water molecule when it isjoined to another amino acid.

As used herein, the term “amino acid analog” refers to a structuralderivative of an amino acid parent compound that often differs from itby a single element.

As used herein, the term “modified amino acid” refers to an amino acidbearing an “R” substituent that does not correspond to one of the twentygenetically coded amino acids.

As used herein, the abbreviations for the genetically encodedL-enantiomeric amino acids are conventional and are as follows: TheD-amino acids are designated by lower case, e.g. D-proline=p, etc.

TABLE 1 Amino Acids One-Letter Symbol Common Abbreviation Alanine A AlaArginine R Arg Asparagine N Asn Aspartic acid D Asp Cysteine C CysGlutamine Q Gln Glutamic acid E Glu Glycine G Gly Histidine H HisIsoleucine I Ile Leucine L Leu Lysine K Lys Phenylalanine F Phe ProlineP Pro Serine S Ser Threonine T Thr Tryptophan W Trp Tyrosine Y TyrValine V Val

Certain amino acid residues in the active agent-conjugate can bereplaced with other amino acid residues without significantlydeleteriously affecting, and in many cases even enhancing, the activityof the peptides. Thus, also contemplated by the preferred embodimentsare altered or mutated forms of the active agent-conjugate wherein atleast one defined amino acid residue in the structure is substitutedwith another amino acid residue or derivative and/or analog thereof. Itwill be recognized that in preferred embodiments, the amino acidsubstitutions are conservative, i.e., the replacing amino acid residuehas physical and chemical properties that are similar to the amino acidresidue being replaced.

For purposes of determining conservative amino acid substitutions, theamino acids can be conveniently classified into two maincategories—hydrophilic and hydrophobic—depending primarily on thephysical-chemical characteristics of the amino acid side chain. Thesetwo main categories can be further classified into subcategories thatmore distinctly define the characteristics of the amino acid sidechains. For example, the class of hydrophilic amino acids can be furthersubdivided into acidic, basic and polar amino acids. The class ofhydrophobic amino acids can be further subdivided into nonpolar andaromatic amino acids. The definitions of the various categories of aminoacids are as follows:

The term “hydrophilic amino acid” refers to an amino acid exhibiting ahydrophobicity of less than zero according to the normalized consensushydrophobicity scale of Eisenberg et al., 1984, J. Mol. Biol.179:125-142. Genetically encoded hydrophilic amino acids include Thr(T), Ser (S), His (H), Glu (E), Asn (N), Gln (Q), Asp (D), Lys (K) andArg (R).

The term “hydrophobic amino acid” refers to an amino acid exhibiting ahydrophobicity of greater than zero according to the normalizedconsensus hydrophobicity scale of Eisenberg, 1984, J. Mol. Biol.179:1.25-142. Genetically encoded hydrophobic amino acids include Pro(P), Ile (I), Phe (F), Val (V), Leu (L), Trp (W), Met (M), Ala (A), Gly(G) and Tyr (Y).

The term “acidic amino acid” refers to a hydrophilic amino acid having aside chain pK value of less than 7. Acidic amino acids typically havenegatively charged side chains at physiological pH due to loss of ahydrogen ion. Genetically encoded acidic amino acids include Glu (E) andAsp (D).

The term “basic amino acid” refers to a hydrophilic amino acid having aside chain pK value of greater than 7. Basic amino acids typically havepositively charged side chains at physiological pH due to associationwith hydronium ion. Genetically encoded basic amino acids include His(H), Arg (R) and Lys (K).

The term “polar amino acid” refers to a hydrophilic amino acid having aside chain that is uncharged at physiological pH, but which has at leastone bond in which the pair of electrons shared in common by two atoms isheld more closely by one of the atoms. Genetically encoded polar aminoacids include Asn (N), Gln (Q) Ser (S) and Thr (T).

The term “nonpolar amino acid” refers to a hydrophobic amino acid havinga side chain that is uncharged at physiological pH and which has bondsin which the pair of electrons shared in common by two atoms isgenerally held equally by each of the two atoms (i.e., the side chain isnot polar). Genetically encoded nonpolar amino acids include Leu (L),Val (V), Ile (I), Met (M), Gly (G) and Ala (A).

The term “aromatic amino acid” refers to a hydrophobic amino acid with aside chain having at least one aromatic or heteroaromatic ring. In someembodiments, the aromatic or heteroaromatic ring may contain one or moresubstituents such as —OH, —SH, —CN, —F, —Cl, —Br, —I, —NO₂, —NO, —NH₂,—NHR, —NRR, —C(O)R, —C(O)OH, —C(O)OR, —C(O)NH₂, —C(O)NHR, —C(O)NRR andthe like where each R is independently (C₁-C₆) alkyl, substituted(C₁-C₆) alkyl, (C₁-C₆) alkenyl, substituted (C₁-C₆) alkenyl, (C₁-C₆)alkynyl, substituted (C₁-C₆) alkynyl, (C₅-C₂₀) aryl, substituted(C₅-C₂₀) aryl, (C₆-C₂₆) alkaryl, substituted (C₆-C₂₆) alkaryl, 5-20membered heteroaryl, substituted 5-20 membered heteroaryl, 6-26 memberedalkheteroaryl or substituted 6-26 membered alkheteroaryl. Geneticallyencoded aromatic amino acids include Phe (F), Tyr (Y) and Trp (W).

The term “aliphatic amino acid” refers to a hydrophobic amino acidhaving an aliphatic hydrocarbon side chain. Genetically encodedaliphatic amino acids include Ala (A), Val (V), Leu (L) and Ile (I).

The amino acid residue Cys (C) is unusual in that it can form disulfidebridges with other Cys (C) residues or other sulfanyl-containing aminoacids. The ability of Cys (C) residues (and other amino acids with —SHcontaining side chains) to exist in a peptide in either the reduced free—SH or oxidized disulfide-bridged form affects whether Cys (C) residuescontribute net hydrophobic or hydrophilic character to a peptide. WhileCys (C) exhibits a hydrophobicity of 0.29 according to the normalizedconsensus scale of Eisenberg (Eisenberg, 1984, supra), it is to beunderstood that for purposes of the preferred embodiments Cys (C) iscategorized as a polar hydrophilic amino acid, notwithstanding thegeneral classifications defined above.

As will be appreciated by those of skill in the art, the above-definedcategories are not mutually exclusive. Thus, amino acids having sidechains exhibiting two or more physical-chemical properties can beincluded in multiple categories. For example, amino acid side chainshaving aromatic moieties that are further substituted with polarsubstituents, such as Tyr (Y), may exhibit both aromatic hydrophobicproperties and polar or hydrophilic properties, and can therefore beincluded in both the aromatic and polar categories. The appropriatecategorization of any amino acid will be apparent to those of skill inthe art, especially in light of the detailed disclosure provided herein.

While the above-defined categories have been exemplified in terms of thegenetically encoded amino acids, the amino acid substitutions need notbe, and in certain embodiments preferably are not, restricted to thegenetically encoded amino acids. In some embodiments, the activeagent-conjugate may contain genetically non-encoded amino acids. Thus,in addition to the naturally occurring genetically encoded amino acids,amino acid residues in the active agent-conjugate may be substitutedwith naturally occurring non-encoded amino acids and synthetic aminoacids.

Certain commonly encountered amino acids which provide usefulsubstitutions for the active agent-conjugates include, but are notlimited to, β-alanine (β-Ala) and other omega-amino acids such as3-aminopropionic acid, 2,3-diaminopropionic acid (Dpr), 4-aminobutyricacid and so forth; α-aminoisobutyric acid (Aib); ε-aminohexanoic acid(Aha); δ-aminovaleric acid (Ava); N-methylglycine or sarcosine (MeGly);ornithine (Orn); citrulline (Cit); t-butylalanine (t-BuA);t-butylglycine (t-BuG); N-methylisoleucine (MeIle); phenylglycine (Phg);cyclohexylalanine (Cha); norleucine (Nle); naphthylalanine (Nal);4-phenylphenylalanine, 4-chlorophenylalanine (Phe(4-Cl));2-fluorophenylalanine (Phe(2-F)); 3-fluorophenylalanine (Phe(3-F));4-fluorophenylalanine (Phe(4-F)); penicillamine (Pen);1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic);β-2-thienylalanine (Thi); methionine sulfoxide (MSO); homoarginine(hArg); N-acetyl lysine (AcLys); 2,4-diaminobutyric acid (Dbu);2,3-diaminobutyric acid (Dab); p-aminophenylalanine (Phe (pNH₂));N-methyl valine (MeVal); homocysteine (hCys), homophenylalanine (hPhe)and homoserine (hSer); hydroxyproline (Hyp), homoproline (hPro),N-methylated amino acids and peptoids (N-substituted glycines).

Other amino acid residues not specifically mentioned herein can bereadily categorized based on their observed physical and chemicalproperties in light of the definitions provided herein.

The classifications of the genetically encoded and common non-encodedamino acids according to the categories defined above are summarized inTable 2, below. It is to be understood that Table 2 is for illustrativepurposes only and does not purport to be an exhaustive list of aminoacid residues and derivatives that can be used to substitute the activeagent-conjugate described herein.

TABLE 2 CLASSIFICATIONS OF COMMONLY ENCOUNTERED AMINO ACIDSClassification Genetically Encoded Non-Genetically Encoded HydrophobicAromatic F, Y, W Phg, Nal, Thi, Tic, Phe (4-Cl), Phe (2-F), Phe (3-F),Phe (4-F), hPhe Nonpolar L, V, I, M, G, A, P t-BuA, t-BuG, MeIle, Nle,MeVal, Cha, McGly, Aib Aliphatic A, V, L, I b-Ala, Dpr, Aib, Aha, MeGly,t-BuA, t-BuG, MeIle, Cha, Nle, MeVal Hydrophilic Acidic D, E Basic H, K,R Dpr, Orn, hArg, Phe(p-NH₂), Dbu, Dab Polar C, Q, N, S, T Cit, AcLys,MSO, bAla, hSer Helix-Breaking P, G D-Pro and other D-amino acids (inL-peptides)

Other amino acid residues not specifically mentioned herein can bereadily categorized based on their observed physical and chemicalproperties in light of the definitions provided herein.

While in most instances, the amino acids of the active agent-conjugatewill be substituted with L-enantiomeric amino acids, the substitutionsare not limited to L-enantiomeric amino acids. In some embodiments, thepeptides may advantageously be composed of at least one D-enantiomericamino acid. Peptides containing such D-amino acids are thought to bemore stable to degradation in the oral cavity, gut or serum than arepeptides composed exclusively of L-amino acids.

Conjugation Methods

One-Step Conjugation

Two-Step Conjugation, Drug Preloaded

Two-Step Conjugation, Drug Added at the Last Step

Examples of One-Step Conjugation

Examples Include, but are not Limited

Additional compounds that can be made according to the Generalprocedures include, but are not limited to, the following:

Examples Include, but are not Limited, the Following

Two-Step Conjugation, Drugs Preloaded

Approach:

Examples Include, but are not Limited to, the Following

Conjugation Method

Examples Include, but are not Limited to the Following

Conjugation Method

Examples Include, but are not Limited to, the Following

Examples Antibody-Drug Conjugation General Conjugation Procedure

To a target antibody, 0.5-50 mgs/mL, in a certain buffet at pH 5.0-9.0,such as PBS, was added 0.5-100 eq of reducing agent such as TCEP andDTT. The reduction was performed at 0-40° C. for 0.5-40 hours withgentle stirring or shaking, and then the reducing agent was removed bycolumn or ultrafiltration. To the partially reduced antibody, 0.5-50mgs/mL, in a certain buffet at pH 5.0-9.0, such as PBS, with 0-30% oforganic co-solvent such as DMA, was added 0.5-10 eq of the activateddrug-linker bearing a dibromo or dichloro-functionality. The reactionwas conducted at 0-40° C. for 0.5-40 hours with gentle stirring orshaking, monitored by HIC-HPLC. The resultant crude ADC productunderwent necessary down-stream steps of desalt, buffetchanges/formulation, and optionally, purification, using thestate-of-art procedures. The final ADC product was characterized byHIC-HPLC, SEC, RP-HPLC, and optionally LC-MS. The average DAR wascalculated by UV absorption and/or MS spectroscopy.

General Synthetic Procedures General Procedure A—HATU Mediated AmideBond Formation

To an acid (1.1 eq with respect to amine) in anhydrous DMF was addedHATU (1 eq with respect to acid) and DIEA (2 eq with respect to acid)and the mixture was stirred at room temperature for 1 minute. Themixture was then added to a solution of amine in DMF and the reactionmixture was stirred at room temperature till the completion of thereaction (monitored by LC/MS). The solvent was removed under reducedpressure and the residue was optionally purified by reverse phase HPLCto give final pure product.

General Procedure B—DIC/HOAt Mediated Amide Bond Formation

To a stirred solution of carboxylic acid (1.1 eq), amine and HOAt (1.1eq) in anhydrous DMF was added DIC (1.1 eq) and the reaction mixture wasstirred at room temperature. Upon completion (monitored by LC/MS), thesolvent was removed under reduced pressure and the residue wasoptionally purified by reverse phase HPLC to give final pure product.

General Procedure C—Removal of Acid Sensitive Protecting Groups (Boc,THP, t-Bu) Using HCl/Dioxane

The acid sensitive protecting groups containing compound was dissolvedin 4N HCl/dioxane and the mixture was stirred at room temperature for 2h. The solution was then concentrated under reduced pressure and theresidue was washed twice with cold ether. Purification was carried outon reverse phase HPLC if necessary.

General Procedure D—Removal of Fmoc Group

The Fmoc containing compound was dissolved in 2-5% piperidine in DMF.The mixture was stirred at room temperature for 1 h. The solvents wereremoved under reduced pressure. Purification was carried out on reversephase HPLC if necessary.

General Procedure E—Reductive Alkylation

An amine was dissolved in DMF and aldehyde (5 eq) was added, followed byaddition of sodium cyanoborohydride (5 eq). HOAc was added to adjust thepH of the reaction mixture to 4-5. The mixture was stirred at roomtemperature till completion (1-4 h, monitored by HPLC). Purification wascarried out on reverse phase HPLC if necessary.

General Procedure F—Saponification—Removal of Me/et from Esters

To a stirred solution of an ester in MeOH was added 1M aq. solution ofLiOH till pH of the mixture was about 13-14 and the reaction mixture wasstirred at room temperature till completion (˜16 h, monitored by HPLC).Citric acid (˜10% aq,) was added to neutralize the reaction and thesolvents were removed under reduced pressure. The crude product wasoptionally purified by RP-HPLC or used directly in the next step.

General Procedure G—Activation of a Hydroxyl/Phenol Group withBis(p-Nitrophenyl)Carbonate

To a stirred solution of an alcohol/phenol in THF/DMF (2/1) was addedbis(p-nitrophenyl) carbonate (3-5 eq), followed by DIEA (2-4 eq) and thereaction mixture was stirred at room temperature until most of thestarting material was consumed. The progress of the reaction wasmonitored by LC/MS. The crude product was optionally purified by flashcolumn chromatography or by precipitation and washing.

General Procedure H—Reaction of an Amine with a Cyclic Anhydride(Glutaric Anhydride or Succinic Anhydride)

An amine containing compound was dissolved in DMF. Glutaric anhydride (3eq) was added, followed by addition of DIEA (4 eq). The reaction mixturewas stirred at room temperature until most of the starting material wasconsumed. The progress of the reaction was monitored by LC/MS. The crudeproduct was purified by RP-HPLC to yield the pure carboxylic acid.

General Procedure I—Formation of Carbamate with p-Nitrophenyl Carbonate(e.g. FmocVC-PAB-PNP)

An amine containing compound was dissolved in DMF and alkyl/arylp-nitrophenyl carbonate (1.5 eq) was added, followed by addition of DIEA(2 eq) and HOBt (cat., 5%). The reaction mixture was stirred at roomtemperature until most of the amine was consumed. The progress of thereaction was monitored by LC/MS. The crude product was optionallypurified by RP-HPLC to yield the pure carbamate.

General Procedure J—Formation of an Activated Ester (e.g. NHS) from anAcid

An acid was dissolved in DCM and DMF was added to aid dissolution ifnecessary. N-hydroxysuccinimide (1.5 eq) was added, followed by EDC.HCl(1.5 eq). The reaction mixture was stirred at room temperature for 1 huntil most of the acid was consumed. The progress of the reaction wasmonitored by RP-HPLC. The mixture was then diluted with DCM and washedsuccessively with citric acid (aq. 10%) and brine. The organic layer wasdried and concentrated to dryness. The crude product was optionallypurified by RP-HPLC or silica gel column chromatography.

Conjugation Method

Conjugation on two Cys residues by forming a cyclic structure

Two-Step Method

One-Step Method

Triazole One Step Method

Triazine One Step Method

Experimental Description Step 1. Drug-Linker Construct Synthesis (-L²-D)Methods of Drug-Linker Construct Synthesis, but No Limited to: Method1-1: Linker and Drug Connected Via a Carbamate Bond Using GeneralProcedure G or I for Activation and Carbamate Formation and GeneralProcedure C, D, or F for Removal of Protective Groups.

Method 1-2: Linker and Drug Connected Via Reductive Alkylation Reaction(General Procedure E)

Method 1-3. Active Molecule Containing a Carboxylic Acid MoietyConnected to an Alkoxyamino Linker Via Formation of Hydoxamate (GeneralProcedure A or B), Followed by Removal of Protective Groups.

For active molecules that are hydoxamic acids, the above method stillcan be employed since the construct will release hydroxamic acid underenzymatic cleavage conditions. The reaction needs to start from itscorresponding carboxylic acid.

Step 2. Introducing Functional Groups to L1-(L2-D)

Method 2. Introduction of o-Phenylenediamine Moiety

The 3-nitro-4-amino benzoic acid was incorporated using standardamidation reaction (General procedure B). The nitro group was reducedusing sodium dithionite (3 eq) in acetonitrile/water to give the desiredo-phenylene diamine. MS found: 1504.8 (M+H)⁺.

Step 3. Introducing the Final Functional Groups Prior to ConjugationMethods of Introduction of Final Reactive Group Prior to ConjugationReaction, but not Limited to: Method 3-1. Formation of DibromomethylQuinoxaline

The o-phenylenediamine compound was dissolved in acetonitrile/water.Dibromomethyl diketone was added. The mixture was stirred at roomtemperature and purified directly by RP-HPLC to give the desiredquinoxaline.

Method 3-2. Formation of Dichlorotriazine

To a stirred and cooled (ice bath) solution of amine compound (12 mg) inDMF (1 mL) was added a solution of cyanuric chloride (10 mg) in THF (0.5mL) and DIEA (5 μL). The reaction mixture was stirred at roomtemperature for 10 mins, and then concentrated under reduced pressure.The residue was purified by RP-HPLC to give the desired dichlorotraizine(11 mg) as a white powder after lyophilization. MS found 1383.9 (M+H)⁺.

Method 3-3. Formation of Dichloromethyltriazole

To a stirred solution of VC-PAB-MMAE (30 mg) in acetonitrile/water (2mL, 6/4, v/v) was added a solution of trizole NHS ester (15 mg) inacetonitrile (0.5 mL) and sat. aq. NaHCO₃ (0.1 mL). The reaction mixturewas purified by RP-HPLC to give the desired product as a white powderafter lyophilization (28 mg). MS found: 1356.9 (M+H)⁺

Example I Synthesis of Compound 10

Scheme I.

Reagents and conditions: i. SOCl₂, EtOH; ii. DEPC, TFA, DCM; iii. TFA,DCM; iv. BrOP, DCM, DIEA; v. TFA, DCM; iv. DIEA, DCM, HOBt.

To a solution of compound 1 (23.4 g, 81.53 mmol) in dry EtOH (200 ml)was added SOCl₂ (100 mL) at 0° C. The mixture was stirred for overnightand the solvent was removed by evaporation in vacuo. The residue wasimmediately used for the next step without further purification. To asolution of compound 2 (81.53 mmol), compound 3 (50 g, 163.1 mmol) indry DMF (150 mL) was added DEPC (15.9 g, 97.8 mmol), TEA (41 g, 0.408mol) at 0° C. The mixture was stirred for 2 h at 0° C. Then the mixturewas stirred overnight at room temperature. Solvent was removed byevaporation in vacuo. The residue was diluted with ethyl acetate-toluene(2:1, 900 ml) and washed with 1M KHSO₄, water, sat. NaHCO₃, and brine.The organic layer was dried and concentrated to give a residue, whichwas purified by column (hexanes:ethyl acetate:DCM=5:1:1) to give 38 g ofcompound 4.

To a solution of Boc-Val-OH (30.6 g, 0.142 mol), compound 5 (from 25 gof compound 4) in DCM (400 mL) was added BrOP (28 g, 70.87 mmol), DIEA(30 g, 0.236 mol) at 0° C. The mixture was shielded from light andstirred for 0.5 h at 0° C. Then the mixture was stirred for 48 h at roomtemperature. The solvent was removed by evaporation in vacuo. Theresidue was diluted with ethyl acetate-toluene (3:1, 900 mL) and washedwith 1M KHSO₄, water, sat. NaHCO₃, and brine. The organic layer wasdried and concentrated to give a residue, which was purified by silicagel column (hexanes:ethyl acetate:DCM=3:1:1) to give 22 g of compound 7.

To a solution of compound 7 (40 g, 66.7 mmol) in THF (600 mL) was addeda mixture of LiOH (14 g, 0.33 mol) in water (300 mL) below 10° C. Themixture was stirred for 5 days at 25° C. THF was removed by evaporation.The aqueous layer was washed with Et₂O (200 mL×3). The aqueous layer wasacidified to pH 2 with 1N HCl at 0° C., the mixture was extracted withethyl acetate and the organic layer was washed with water and brine. Theorganic layer was dried and concentrated to give a residue, which waspurified by Prep-HPLC to give 14 g of compound 8.

To a solution of compound 8 (3 g) in DCM (100 mL) was added compound 9(3 g, prepared according to General procedure J from Boc-N-Me-Val-OHusing EDC and pentafluorophenol). DIEA (2.6 mL) was added, followed byHOBt (cat. 100 mg) and the reaction mixture was stirred at roomtemperature for 16 h. The solvents were removed under reduced pressureand the residue was purified on a silica gel column to give compound 10as a white powder (3.1 g). MS m/z Calcd for C₃₅H₆₄N₄O₉ 684.5. Found707.6 ([M+Na]⁺).

Example II-1 Synthesis of Compound 13

Scheme II-1.

Reagents and conditions: i. DIC/HOAt, DMF, rt, 16 h; ii. HCl/Dioxane

The amino acid sulfonamide derivatives 11 were synthesized according topreviously reported procedure (ARKIVOC 2004 (xii) 14-22, or WO2007146695) using Boc protected amino acid and cyclopropyl/methylsulfonamide, followed by removal of Boc (General procedure C)

Compound 13 was synthesized using the general procedures described aboveas following: DIC/HOAt mediated amide bond formation (General procedureB) between Boc-N-Me-Val-Val-Dil-Dap-OH (compound 1) and amine 11,followed by removal of Boc (General procedure C). The final compound waspurified by reverse phase HPLC to give compound 13 as a white powderafter lyophilization. MS m/z Calcd for C₄₂H₇₀N₆O₉S, 834.5. Found 835.6([M+H]⁺).

Example II-2 Synthesis of Compounds 16

Scheme II-2.

Reagents and conditions: i. DIC/HOAt, DMF, rt, 16 h; ii. HCl/Dioxane

The amino acid sulfonamide derivatives 14 were synthesized according topreviously reported procedure (ARKIVOC 2004 (xii) 14-22, or WO2007146695) using Boc protected amino acid and cyclopropyl/methylsulfonamide, followed by removal of Boc (General procedure C).

Compound 16 was synthesized using the general procedures described aboveas following: DIC/HOAt mediated amide bond formation (General procedureB) between Boc-N-Me-Val-Val-Dil-Dap-OH (compound 10) and amine 14,followed by removal of Boc (General procedure C). The final compound waspurified by reverse phase HPLC to give compound 16 as a white powderafter lyophilization. MS m/z Calcd for C₄₁H₇₀N₆O₁₀S, 838.5. Found 839.6([M+H]⁺).

Example II-3 Synthesis of Compound 20

Scheme IIc.

Reagents and conditions: i. bis(nitrophenyl) carbonate, DIEA, THF/DMF,r.t.; ii. 6-aminohexanoic acid, NaHCO₃ (aq.); iii. HCl/Dixoane (4N); iv.HCHO, NaCNBH₃, DMF, HOAc.

The phenol 16 (1 mmol) was treated with 3 eq ofbis(p-nitrophenyl)carbonate to form the activated carbonate 17 (generalprocedure G). The crude product was used directly without furtherpurification. 6-Aminohexanoic acid (5 eq) was dissolved in sat. aq.NaHCO₃ (5 mL) and the solution was added. The reaction mixture wasstirred at room temperature for 16 h. Citric acid (aq. 10%) was added toacidify the reaction (pH=4-5) and then diluted with EtOAc (150 mL).Organic layer was dried (over Na₂SO₄) and concentrated to give the crudeproduct 18 which underwent the following procedures: removal of Boc(General procedure C), reductive alkylation using HCHO. The finalproduct was purified by RP-HPLC to give compound 20 as a white powderafter lyophilization. MS m/z Calcd for C₄₈H₈₁N₇O₁₃S, 995.6. Found 996.4([M+H]⁺).

Example III Synthesis of Alkoxylamine Linkers 24, 25, 26, and 27

ID Structure 24

25

26

27

Scheme III.

Reagents and conditions: i. SOCl₂, THF, 1 h; ii. N-hydroxyphthalimide,NaHCO₃, DMF, rt, 48 h; iii. NH₂NH₂.H₂O, HOAc, DMF.

Example III-1 Synthesis of Compound 24

To a stirred solution of Fmoc-VA-PAB (21) (Bioconjugate Chem., 2002, 13,855-859) (9 g, 15 mmol) in THF (200 mL) was added thionyl chloride (18mmol) dropwise. After the addition was complete, the reaction mixturewas stirred at room temperature for 1 h. TLC analysis (ethylacetate/hexane, 1/1, v/v) showed the completion of the reaction. Thesolvents were removed under reduced pressure and the residue was washedwith hexanes (100 mL) to give compound 22 as a slightly yellowish solid(8.8 g).

Compound 22 (6.2 g, 10 mmol) was dissolved in anhydrous DMF (100 mL).N-Hydroxy-phthalimide (3.2 g, 20 mmol) was added, followed by solidNaHCO₃ (3.4 g, 40 mmol). The reaction mixture was stirred at roomtemperature for 48 h. TLC analysis showed that most of compound 61 wasconsumed. The reaction was then diluted with ethyl acetate (500 mL) andwashed successively with sat. aq. NaHCO₃ (3×200 mL) and brine (200 mL).The organic layer was dried and concentrated to give compound 23 as atan solid, which was used directly without further purification.

The crude compound 23 from previous step was dissolved in DMF (100 mL).HOAc (6 mL) was added, followed by hydrazine hydrate (5 mL). Thereaction was stirred at room temperature for 1 h. LC/MS showed thecompletion of the reaction. The reaction mixture was then poured into abeaker containing 1 L of water under stirring. The precipitated solidwas collected via filtration and washed twice with water to givecompound 24 as a white solid (purity >85%, can be used directly). Purecompound 63 was obtained after RP-HPLC purification. MS m/z Calcd forC₃₀H₃₄N₄O₅ 530.3. Found 531.4 ([M+H]⁺).

Example III-2 Synthesis of Compound 25

Compound 25 was synthesized starting from compound Fmoc-VC-PAB(Bioconjugate Chem., 2002, 13, 855-859) using the procedures describedabove for the synthesis of compound 25. MS m/z Calcd for C₃₃H₄₀N₆O₆616.3. Found 617.5 ([M+H]⁺).

Example III-3 Synthesis of Compound 26

Compound 26 was synthesized starting from compound Fmoc-A-PAB(synthesized according to the procedure reported: Bioconjugate Chem.,2002, 13, 855-859) using the procedures described above for thesynthesis of compound 26. MS m/z Calcd for C₂₅H₂₅N₃O₄ 431.2. Found 432.6([M+H]⁺).

Example III-4 Synthesis of Compound 27

Compound 27 was synthesized starting from compound Fmoc-Ahx-PAB usingthe procedures described above for the synthesis of compound 27. MS m/zCalcd for C₂₈H₃₁N₃O₄ 473.2. Found 474.3 ([M+H]⁺).

Example IV Synthesis of -L1-(L2-D)

Scheme IV.

Reagents and conditions: i. DIC, HOAt, DMF, r.t.; ii. Piperidine, DMF;iii. HATU, DIEA, DMF; iv. Na₂S₂O4, MeCN, H₂O, pH=6.

Example IV-1 Synthesis of Compound 32

Compound 32 was synthesized using the general procedures described aboveas following: DIC/HOAt mediated amide bond formation (General procedureB) between Auristatin F and compound 26, followed by removal of Fmoc(General procedure D), reaction with acid 30 (General procedure A), andreduction of the nitro group to amino group.

The nitro compound 31 (32 mg) was dissolved in acetonitrile/water (6/4,v/v) and a solution of sodium dithionite (1M in water, 0.2 mL) wasadded. The pH of the reaction was controlled at 6-6.5 by addition of MESbuffer. The mixture was stirred at room temperature for 2 h and purifieddirectly by RP-HPLC to give the desired product as a white solid afterlyophilization (18 mg). MS found 1319.0 (M+H)⁺.

Example V Introduction of the Final Functional Group Prior toConjugation Reaction

ID Structure of —L¹—(L²—D) MS 34

1713.0 40

1526.4 36

1383.9 39

1356.9 41

1281.2 42

1424.5 43

1249.5 44

1621.7 45

1572.7 46

1288.3 47

1510.8 48

1250.6 49

1195.4 50

1020.4

Example V-1 Formation of Dibromomethyl Quinoxaline

The o-phenylenediamine compound 32 (12 mg) was dissolved inacetonitrile/water (1 mL). Dibromomethyl diketone (10 mg) was added. Themixture was stirred at room temperature and purified directly by RP-HPLCto give the desired quinoxaline 40 as a white powder (12 mg) afterlyophilization. MS found 1526.4 (M+H)⁺.

Compound 34, 42, 43, 44, 45, 46, and 47 were synthesized from thecorresponding o-phenylenediamines using the synthetic proceduredescribed above for the synthesis of compound 40.

Example V-2 Formation of Dichlorotriazine

To a stirred and cooled solution of amine compound 35 (12 mg) in DMF (1mL) was added a solution of cyanuric chloride (10 mg) in THF (0.5 mL)and DIEA (5 μL). The reaction mixture was stirred at room temperaturefor 10 mins, and then concentrated under reduced pressure. The residuewas purified by RP-HPLC to give the desired dichlorotraizine 36 (11 mg)as a white powder after lyophilization. MS found 1383.9 (M+H)⁺.

Compound 41 was synthesized using the same procedure described above forthe synthesis of compound 36. MS found 1281.2 (M+H)⁺.

Example V-3 Formation of Dichloromethyltriazole

To a stirred solution of VC-PAB-MMAE (38, 30 mg) in acetonitrile/water(2 mL, 6/4, v/v) was added a solution of trizole NHS ester (37, 15 mg)in acetonitrile (0.5 mL) and sat. aq. NaHCO₃ (0.1 mL). The reactionmixture was purified by RP-HPLC to give the desired product 39 as awhite powder after lyophilization (28 mg). MS found: 1356.9 (M+H)⁺.

Example V-4 Synthesis of Compound 50

Scheme V-4.

Reagents and conditions: i. Fmoc-Ahx-OH (aminohexanoic acid), DIC, DCM,Py, rt, 48 h; ii. Piperidine, DMF; iii. DIC, HOBt; iv. Dibromomethyldiketone.

Fmoc-Ahx-OH (353 mg) was suspended in anhydrous DCM (5 mL) and DIC (0.5mmol) was added. The mixture was stirred at room temperature for 1 h.Maytansinol (compound 51, 56 mg) was added to the mixture, followed byaddition of 0.5 mL of pyridine. The reaction mixture was stirred at roomtemperature for 48 h. Solvents were removed under reduced pressure andthe residue was purified by column chromatography (silica gel,EA/hexanes) to give compound 52 as a white solid (45 mg) which wastreated with piperidine to remove Fmoc (General procedure D), followedby amidation reaction with 3,4-diaminobenzoic acid (1 eq) using DIC/HOBt(General procedure B, using HOBt instead of HOAt) to give compound 54(22 mg) as a slightly yellowish powder after HPLC purification andlyophilization. Compound 54 was then converted to the desired compound50 using the same procedure described above for the synthesis ofcompound 40. MS found 1020.4 (M+H)⁺.

Antibody-Drug Conjugation

General Conjugation Procedure

To a target antibody, 0.5-50 mgs/mL, in a certain buffet at pH 5.0-9.0,such as PBS, was added 0.5-100 eq of reducing agent such as TCEP andDTT. The reduction was performed at 0-40° C. for 0.5-40 hours withgentle stirring or shaking, and then the reducing agent was removed bycolumn or ultrafiltration. To the partially reduced antibody, 0.5-50mgs/mL, in a certain buffet at pH 5.0-9.0, such as PBS, with 0-30% oforganic co-solvent such as DMA, was added 0.5-10 eq of the activateddrug-linker bearing a dibromo or dichloro-functionality. The reactionwas conducted at 0-40° C. for 0.5-40 hours with gentle stirring orshaking, monitored by HIC-HPLC. The resultant crude ADC productunderwent necessary down-stream steps of desalt, buffetchanges/formulation, and optionally, purification, using thestate-of-art procedures. The final ADC product was characterized byHIC-HPLC, SEC, RP-HPLC, and optionally LC-MS. The average DAR wascalculated by UV absorption and/or MS spectroscopy.

Trastuzumab and compound 34 were combined in various drug/antibodyratios under as described in the General Conjugation Procedure.Trastuzumab was incubated with 10 mM TCEP stock solution at 37° C. for0.5 to 40 hours. After reduction, the excess of TCEP was removed by agel filtration column. The reduced trastuzumab was then mixed withcompound 34 in drug/antibody ratio range from 1 to 10 at 0 to 40° C.After overnight reaction, excess compound 34 was removed fromTrastuzumab-c-34 conjugate by a gel filtration column. PurifiedTrastuzumab-c-34 conjugates stored at 4 to −20° C. and to be used for invitro and in vivo tests.

The product of the conjugation reaction was analyzed using HIC-HPLCunder conditions: HPLC Column: Tosoh TSKgel Butyl-NPR, 4.6 mm×3.5 cm,2.5 mm; Buffer A: 20 mM sodium phosphate, 1.5 M Ammonium Sulfate, pH7.0; Buffer B: 20 mM sodium phosphate, 25% v/v isopronal, pH 7.0; flowrate: 1 mL/min; Gradient: 10 min 10% Buffer B to 80% Buffer B, 4 min100% Buffer B; 20 μL sample.

FIG. 1 shows the HIC-HPLC chromatogram product of reacting drug andantibody at a drug/antibody ratio of 5.5:1, where DAR represents thenumber of drugs conjugated per antibody. FIG. 2 shows the HIC-HPLCchromatogram product of reacting drug and antibody at a drug/antibodyratio of 6:1, where DAR represents the number of drugs conjugated perantibody. FIG. 3 shows the HIC-HPLC chromatogram product of reactingdrug and antibody at a drug/antibody ratio of 1:6.5, where DARrepresents the number of drugs conjugated per antibody. FIG. 4 shows theHIC-HPLC chromatogram product of reacting drug and antibody at adrug/antibody ratio of 1:4, where DAR represents the number of drugsconjugated per antibody. In overlay, FIG. 4 further shows HIC-HPLCchromatograms for individually isolated fractions of DAR 1, DAR 2, DAR3, DAR 4 and DAR 5 peaks purified using FPLC. FIG. 5 shows HIC-HPLCchromatograms for each FPLC fraction, and, inset, an overlay of thepeaks to demonstrate relative purity of each peak.

The purified fractions depicted in FIGS. 4 and 5 were further analyzedby reduced SDS-PAGE. The ADC samples were reduced with 20 mM DTT andloaded onto a NuPAGE 4-12% Bis-Tris Gel (Life Technology). The resultsare shown in FIG. 6, where the abbreviations are as follows:

H-antibody heavy chain

L-antibody light chain

HH-heavy chain dimer

HL-heavy and light chain dimer

HHL-heavy-heavy and light chain trimer

IgG-two heavy-two light chain tetramer

In Vitro Cytotoxicity Experiment

The antibody drug conjugates were the analyzed for cytotoxicity. Thecell lines used were SK-BR-3 human breast adenocarcinoma (HER2 triplepositive), HCC1954 human Ductal Carcinoma (HER2 triple positive). Thesecells were available from ATCC. SK-BR-3 cells were grown in McCoy's 5Amedium (Caisson Labs, North Logan, Utah) supplemented with 10% fetalbovine serum. HCC1954 cells were grown in RPMI-1640 medium (CaissonLabs, North Logan, Utah) supplemented with 10% fetal bovine serum.SK-BR-3 cells were plated in 96-well plates at approximately 7,500cells/well, and HCC1954 cells were plated in 96-well plates atapproximately 20,000 cells/well. Antibody-drug conjugates were added induplicates in the same day. After 72 hour incubation at 37° C.,CellTiter-Glo (Promega, Madison, Wis.) were added and cell viability wasdetermined as describe by the manufacture's protocol. The percentviability was determined as following:

% Viability=Average Luminescence Value of the duplicates(treatedwells)/Average Luminescence Value of the untreated wells

FIG. 7 shows viability of SK-BR-3 cells for seven samples, ADC_C1 (DAR1from FIGS. 4 and 5), ADC_C2 (DAR2 from FIGS. 4 and 5), ADC_C3 (DAR3 fromFIGS. 4 and 5), ADC_C4 (DAR4 from FIGS. 4 and 5), ADC_(—)5 (DAR5 fromFIGS. 4 and 5), SeaGen (ADC prepared following published SeattleGenetics method), and T-DM1 (ADC prepared following published Immunogenmethod).

FIG. 8 shows viability of HCC1954 cells for seven samples, ADC_C1 (DAR1from FIGS. 4 and 5), ADC_C2 (DAR2 from FIGS. 4 and 5), ADC_C3 (DAR3 fromFIGS. 4 and 5), ADC_C4 (DAR4 from FIGS. 4 and 5), ADC_C5 (DAR5 fromFIGS. 4 and 5), ADC_VC-M MAE was prepared following published SeattleGenetics method), and T-DM1 (ADC prepared following published Immunogenmethod).

FIG. 10 shows viability of SK-BR-3 cells for four samples, both ADC_C6(DAR2.4) and ADC_C7 (DAR3.1) were prepared using the General ConjugationProcedure and purified by a gel filtration column. SeaGen (ADC preparedfollowing published Seattle Genetics method), and T-DM1 (ADC preparedfollowing published Immunogen method).

FIG. 11 shows viability of HCC1954 cells for four samples, ADC_C6(DAR2.4 from FIG. 10), ADC_C7 (DAR3.1 from FIG. 10), ADC_VC-M MAE wasprepared following published Seattle Genetics method), and T-DM1 (ADCprepared following published Immunogen method).

Antibody-Drug Conjugation

Trastuzumab and compound 42 were combined in various drug/antibodyratios under conditions where Trastuzumab was incubated with 10 mM TCEPstock solution at 37° C. for 0.5 to 40 hours. After reduction, theexcess of TCEP was removed by a gel filtration column. The reducedTrastuzumab was then mixed with compound 42 in drug/antibody ratio rangefrom 1 to 10 at 0 to 40° C. After overnight reaction, excess compound 42was removed from Trastuzumab-c-34 conjugate by a gel filtration column.Purified Trastuzumab-c-42 conjugates stored at 4 to −20° C. and to beused for in vitro and in vivo tests.

Trastuzumab and compound 40 were combined in various drug/antibodyratios under conditions where Trastuzumab was incubated with 10 mM TCEPstock solution at 37° C. for 0.5 to 40 hours. After reduction, theexcess of TCEP was removed by a gel filtration column. The reducedTrastuzumab was then mixed with compound 40 in drug/antibody ratio rangefrom 1 to 10 at 0 to 40° C. After overnight reaction, excess compound 40was removed from Trastuzumab-c-34 conjugate by a gel filtration column.Purified Trastuzumab-c-40 conjugates stored at 4 to −20° C. and to beused for in vitro and in vivo tests.

The product of the conjugation reaction was analyzed using reducedSDS-PAGE. The ADC samples were reduced with 20 mM DTT and loaded onto aNuPAGE 4-12% Bis-Tris Gel (Life Technology). The results are shown inFIG. 9, where the abbreviations are as follows:

H-antibody heavy chain

L+vc-MMAE-antibody light chain-vc-MMAE

L-antibody light chain

HH-heavy chain dimer

HL-heavy and light chain dimer

HHL-heavy-heavy and light chain trimer

IgG-two heavy-two light chain tetramer

cL-Duo3: Trastuzumab—compound 42 conjugate

cL-Duo6: Trastuzumab—compound 40 conjugate

In Vitro Cytotoxicity Experiment

The Trastuzumab—compound 42 and Trastuzumab—compound 42 conjugates werethe analyzed for cytotoxicity. The cytotoxicity methods were performedas described above.

FIG. 12 shows viability of SK-BR-3 cells for four samples,Trastuzumab-c-Duo3 (Trastuzumab—compound 42 conjugate),Trastuzumab—c-Duo6 (Trastuzumab—compound 40 conjugate),Trastuzumab-vc-MMAE (prepared following published Seattle Geneticsmethod), and Trastuzumab—c-MMAE (Trastuzumab—compound 34 conjugate,prepared following published Seattle Genetics method).

1. (canceled)
 2. An active agent-conjugate composition comprisingFormula Ia:

or a pharmaceutically acceptable salt thereof, wherein: the A-componentis a targeting moiety; the E-component is an optionally substitutedheteroaryl or an optionally substituted heterocyclyl; L³ is anoptionally substituted C₁-C₆ alkyl, or L³ is null, when L³ is null thesulfur is directly connected to the E-component; L⁴ is an optionallysubstituted C₁-C₆ alkyl, or L⁴ is null, when L⁴ is null the sulfur isdirectly connected to the E-component; each D is independently selected,wherein each D includes an active agent; each L² is independently alinker, wherein at least one L² links to L¹; and n is 1, 2, 3, 4, 5, 6,7, 8, 9, or
 10. 3. The active agent-conjugate of claim 2, wherein theE-component includes a fragment selected from the group consisting of:


4. The active agent-conjugate of claim 2, wherein L³ is —(CH₂)—; and L⁴is —(CH₂)—.
 5. The active agent-conjugate of claim 2, wherein L³ isnull; and L⁴ is null.
 6. The active agent-conjugate of claim 2, wherein:

is selected from the group consisting of:


7. The active agent-conjugate of claim 2, wherein said an active agentis independently selected from the group consisting of tubulin binders,DNA alkylators, DNA intercalator, enzyme inhibitors, immune modulators,peptides, and nucleotides.
 8. The active agent-conjugate of claim 2,wherein the structure of Formula Ia has a structure selected from thegroup consisting of:

9-25. (canceled)
 26. The active agent-conjugate of claim 2, wherein L²is selected from the group consisting of —(CH₂)_(n)—, —(CH₂CH₂O)_(n)—,Val-Cit-PAB, Val-Ala-PAB, Val-Lys(Ac)-PAB, Phe-Lys-PAB, Phe-Lys(Ac)-PAB,D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn-PAB, Ala-PAB, PAB, peptide,oligosaccharide, —(CH₂)_(n)—, —(CH₂CH₂O)_(n)—, Val-Cit-PAB, Val-Ala-PAB,Val-Lys(Ac)-PAB, Phe-Lys-PAB, Phe-Lys(Ac)-PAB, D-Val-Leu-Lys,Gly-Gly-Arg, Ala-Ala-Asn-PAB, Ala-PAB, PAB, and combinations thereof.27-29. (canceled)
 30. The active agent-conjugate of claim 2, wherein L¹a 4-carbon bridge and at least two sulfur atoms. 31-47. (canceled) 48.The active agent-conjugate of claim 2, wherein the A component isselected from the group consisting of a monoclonal antibody (mAB), anantibody fragment, surrogate, or variant, a protein ligand, a proteinscaffold, a peptide, and a small molecule ligand. 49-52. (canceled) 53.The active agent-conjugate of claim 2, wherein the A component comprisesat least one modified L-Alanine residue.
 54. (canceled)
 55. The activeagent-conjugate of claim 2, wherein at least one L² is selected from thegroup consisting of —(CH₂)_(n)—, —(CH₂CH₂O)_(n)—, Val-Cit-PAB,Val-Ala-PAB, Val-Lys(Ac)-PAB Phe-Lys-PAB, Phe-Lys(Ac)-PAB,D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn-PAB, Ala-PAB, PAB, a peptide, anoligosaccharide, —(CH₂)_(n)—, —(CH₂CH₂O)_(n)—, Val-Cit-PAB, Val-Ala-PAB,Val-Lys(Ac)-PAB, Phe-Lys-PAB, Phe-Lys(Ac)-PAB, D-Val-Leu-Lys,Gly-Gly-Arg, Ala-Ala-Asn-PAB, Ala-PAB, PAB and combinations thereof.56-58. (canceled)